Identification And Pathogenicity Analysis Of NR5A1 Variants In Patients With Disorders Of Sex Development

Background: Disorders of sex development?DSD?are congenital conditions in which chromosomal,gonadal or anatomical sex is atypical.The prevalence of genital ambiguity at birth is approximately 1:1,000.Among 46,XY DSD patients,up to 70%lack accurate clinical diagnosis.Approximately 8-15% of all cases of 46,XY DSD are caused by variants involving the gene NR5A1.Different individuals with the same mutation in the NR5A1 gene have different clinical phenotypes.It is difficult to determine the correlation between genotype and phenotype.Subjects and methods: In this paper,a total of sixty-two patients from fifty-nine46,XY DSD families without AR and SRD5A2 variants were selected as the research objects.?1?A total of 164 genes were captured and sequenced in this study.The variants were filtered against four public databases?1000 Genomes Project,GnomAD database,ESP6500 database,ExAC database?and in-house databases.Focus on the NR5A1 gene,and Sanger sequencing was used to validate all candidate pathogenic variants and variant segregation within each patient's family.?2?Possible deleterious effect of each variant on protein structure/function was predicted by SIFT,Polyphen2,SWISS-MODEL and Mutation Taster.?3?Construction of wild type and mutant plasmids of NR5A1 gene for functional study in vitro.The effect of NR5A1 gene mutation on protein expression was studied by Western Blot experiment,the effect of NR5A1 gene mutation on protein localization was studied by ICC experiment and the effect of NR5A1 gene mutation on transcriptional activity was studied by TESCO luciferase assay.Results:?1?Among fifty-nine 46,XY DSD families without AR and SRD5A2 variants,fourteen variants in NR5A1 genes were identified in fourteen unrelated families,including eight heterozygote missense variants;two heterozygote frameshift variants;two heterozygote nonsense variants;one heterozygote nonframeshift deletion-insertion variant and one homozygous missense variant.We achieve a diagnostic rate of approximately 23.72% in NR5A1 variants in 46,XY DSD.?2?Western Blot assay showed that the expression levels of p.Arg69 Gly,p.Thr48 Pro,p.Gly26 Arg,p.Cys55 Gly,p.Arg69 His,p.Ala340 Thr,p.LysVal19₂0delinsLeu mutant proteins were severely lower than wild-type protein;the expression levels of p.Gly328 Arg,p.Asp293 Asn andp.Leu325 Pro did not achieve significant change;variants p.Ser342 X,p.Gln299Argfs*35,p.Cys412 X,and p.Leu361Profs*25 resulted in the production of truncated proteins.?3?ICC assay showed that p.Arg69 His,p.Ser342 X,p.Gln299Argfs*35,p.Cys412 X,p.Leu361Profs*25 and p.Gly26 Arg variants were express not only in the nucleus but also in the cytoplasm,other variants were predominantly localized in the nucleus.?4?TESCO luciferase assay showed that whether it is a single transfection of the NR5A1 plasmid or co-transfection with a SRY or SOX9 plasmid,the relative luciferase activity of p.LysVal19₂0delinsLeu,p.Thr48 Pro,p.Cys55 Gly,p.Arg69 Gly,p.Gln299Arfs*35,p.Leu325 Pro,p.Ser342 X,p.Gly26 Arg,p.Arg69 His,p.Cys412 X and p.Leu361Profs*25variants was significantly decreased;the relative luciferase activity of other variants was not significantly decreased.Conclusion:?1?Fourteen variants in NR5A1 genes were identified in fourteen unrelated families,including nine novel variants.We achieve a diagnostic rate of approximately 23.72% in NR5A1 variants in 46,XY DSD.?2?In vitro functional experiments showed that twelve variants p.LysVal19₂0delinsLeu,p.Thr48 Pro,p.Cys55 Gly,p.Arg69 Gly,p.Gln299Arfs*35,p.Leu325 Pro,p.Ser342 X,p.Gly26 Arg,p.Arg69 His,p.Cys412 X,p.Leu361Profs*25 and p.Ala340 Thr affected protein function;variants p.Asp293 Asn and p.Gly328 Arg did not affect protein function.